MOLECULAR CHARACTERIZATION OF EGGPLANT (Solanum spp.)

 MOLECULAR CHARACTERIZATION OF EGGPLANT (Solanum spp.)

A study was conducted to provide preliminary information on the genetic characterization of eggplant which would contribute to the knowledge of eggplant diversity studies in Nigeria. The DNA was extracted from young leaves of eggplant accession NGB00247 using a plant extraction kit and the NanoDrop spectrophotometer was used to determine the purity and concentration of the DNA which was of good quality. The DNA was amplified using the polymerase chain reaction with ribulose-1, 5-bisphosphate carboxylase (RBC) as the molecular marker. The amplified DNA was sequenced and aligned using the Basic Local Alignment Search Tool (BLAST) nucleotide in the global database of the National Centre for Biotechnology Information (NCBI). The results indicated that the eggplant accession NGB00247 was 99% identical to several Solanum spp due to the region that was sequenced among which are Solanum dasyphyllum and Solanum macrocarpon. With this result, the eggplant accession NGB00247 is likely an intermediate form of the hybridization of Solanum dasyphyllum and Solanum macrocarpon. Preliminary information on the genetic characterization which would contribute to the knowledge of the diversity studies of crops in Nigeria can be gotten through molecular markers.

INTRODUCTION AND LITERATURE REVIEW

1.1 BACKGROUND TO THE STUDY

The eggplant is also known as aubergine or brinjal is a vegetable and a fruit crop, which has been cultivated for centuries in Asia, Africa, and Europe, and is currently a crop species of global importance. The name eggplant has been derived from the shape of the fruit of some varieties, which are white and shaped similarly to chicken eggs. It is also called aubergine, a French word in Europe. The crop originated from South-East Asia (Lester and Hasan, 1991) and belongs to the Solanaceae family (Table 1). Cultivated eggplant and its related species belong to the genus Solanum, which has over 1,500 species (Royal Botanic Gardens and Domain Trust, 2008) and the subgenus Leptostemonum, the largest subgenus comprising nearly one-third of the species within the genus Solanum (Frary et al., 2007). It is a diploid specie 2n=24 with a basic chromosome number of 12 (Doganlar et al., 2002a). The cultivated eggplant was originally described by Carl Linnaeus in his book, Species Plantarum in 1753 (Jarvis 2007; Furini and Wunder, 2004). He described the two important species, S.incanum L. and S.melongena L. which are considered as the cornerstones of the “eggplant complex” (Mace et al., 1999). The genus Solanum has over 1,500 species (Frary et al., 2007). Basically, among the commonly reported species of the genus, Solanum is S.incanum, S.scarbrum, S.dasyphyllum, S.erianthum (Agnieszka et al., 2007; Osei et al., 2010; Knapp et al., 2013; Mariola et al., 2014; Sifau et al., 2014).

The eggplant is a delicate, tropical perennial often cultivated as a tender or half-hardy annual in temperate climates. The stem is often spiny; the flower is white to purple with a five-lobed corolla and yellow stamens. It is egg-shaped, glossy, purple which has white flesh with a meaty texture. Botanically it is classified as a berry of which the fruit contains numerous small, soft seeds that are edible but taste bitter because they contain nicotinoid alkaloids.

Table 1.1: Taxonomic and botanical description of eggplant

Kingdom: Plantae

Division: Angiosperms

Class: Eudicots

Subclass: Asterids

Order: Solanales

Family: Solanaceae

Genus: Solanum

Subgenus: Leptostemonum

Molecular characterization is a fraction of molecular diversity, molecular diversity was a concept introduced by Campbell (2003) and it is referred to as the variation that occurs in DNA sequences within a plant population. It entails the study of genetic or molecular components such as DNA, RNA, and proteins. Characterization is the description of a character, quality of an individual (Miriam-Webster, 1991). Characterization can also be used to mean differentiate or distinguish; this could mean to separate into classes or categories. In genetic terms characterization, characterization means the detection of variation in the DNA sequence, specific genes, or modifying factors (de Vicient et al., 2005). The genetic markers commonly used are morphological markers, isozyme markers, and DNA markers commonly referred to as molecular markers. The study of genetic characterization with the use of molecular markers and molecular methods is known as molecular characterization (de Vicente et al., 2005).  Molecular characterization helps in providing information about the genetic makeup of accessions and helps in decision making of conservation, it helps in determining the breeding behavior of species and helps to know reproductive success and existence of gene flow; that is the movement of alleles within and between populations of the same or related species and its consequences. (Papa and Gepts, 2003). Also, information from molecular markers or DNA sequences offers a good base for better breeding and conservative approaches.

Molecular markers are useful tools for assaying genetic variation and provide an efficient means to link phenotypic and genotypic variations (Varshney et al., 2005). They are also used to determine alleles and it is very important in genetic diversity in the gene pool. There are different ranges of maker types, this is dependent on polymerase chain reaction (PCR).  The importance of molecular makers includes identification of cultivars and species, seed lot purity testing, gene mapping, and gene linkage analysis in association with Quantitative traits Loci (QTL).

There are different types of molecular markers, which are classified as follows: hybridization-based markers, sequenced-based markers and Polymerase chain reaction (PCR) based markers. The PCR-based marker commonly used is the simple sequence repeats (SSR) markers. The discovery of SSR markers has significantly contributed to the conservation of plants and animals (Yu et al., 2000). For plants, the Consortium for the Barcode of Life (CBOL) Plant Working group recommended the plastid DNA (ptDNA) genes rbcl (ribulose-1, 5-bisphosphate carboxylase) and matK (Megakaryocyte-associated tyrosine kinase) as standard DNA barcode markers, based on the availability of universal primers and the high level of taxonomic resolution (Bell et al., 2017). Recent studies using the standard rbcl+matK barcode for floras of moderate phylogenetic dispersion have shown that up to 92% of the species can be distinguished (Bell et al., 2017).

1.2 AIM AND OBJECTIVES

The aim of this study is to provide preliminary information on the genetic characterization of eggplants which would contribute to the knowledge of eggplants diversity studies in Nigeria.

The specific objectives are:

1. To genetically characterize an eggplant accession using ribulose-1, 5-bisphosphate carboxylase (RBC) molecular marker.
2. To compare the eggplant variety’s DNA sequence with the available eggplant sequences in the global database.
3. To identify the eggplant accession.

1.3 LITERATURE REVIEW

1.3.1 ORIGIN AND SPREAD OF EGGPLANT

Eggplant originated from South-East Asia (Lester and Hasan, 1991). Its origin is considered to be India where it continues to grow wild. This crop has been cultivated throughout India and China for more than 1500 years (Trujillo, 2003). The Spaniards carried it with them to the New World and, by the early 1800s, both white and purple varieties could be found in American gardens. As trade routes opened, eggplant was introduced to Europe by the Arabs and transported to Africa by the Persians (Trujillo, 2003).

1.3.2 NUTRITIONAL IMPORTANCE OF EGGPLANT

Eggplant is an important food-security crop worldwide (Fowler et al., 2003; FAO, 2009). The fruit has nominal nutritional value and are fried, stewed, marinated, and prepared in other ways. A serving of 1/2 cup, cubed, boiled eggplant provides 13 calories, 0.1g fat, no cholesterol, 2mg sodium, 0.4g protein, 3.2g carbohydrate, 119g potassium, and other minerals. It is low in calories and dieters can actually snack on garden eggs in-between meals. Eggplant fruits are valuable vegetables for canning industries of garden egg paste and other products. The garden eggplant with its bitter taste and spongy texture could really make an amazing pot of stew with a nice aroma. The eggplant’s natural ability to absorb makes it a nutritional asset to the foods it is prepared with.

1.3.3 CULTURAL IMPORTANCE

According to the American Horticultural Society Encyclopedia of Gardening – Vegetables stated that “a 5th Century Chinese book contains one of the oldest references to eggplant. A black dye was made from the plant, and ladies of fashion used it to stain their teeth – which, when polished, gleamed like metal.” In China, as part of her “bride price,” a woman must have at least 12 eggplant recipes prior to her wedding day. In Turkey, “imam bayeldi,” a tasty treat of stuffed eggplant simmered in olive oil is said to have made a religious leader swoon in ecstasy. When first introduced in Italy, people believed that anyone who ate the “mad apple” was sure to go insane. It was originally domesticated from wild nightshade species and has been cultivated for centuries in Asia, Africa, and Europe and near East. Because of the plant relationship with various nightshades, the fruit was at a point believed to be extremely poisonous. The flowers and leaves can be poisonous if consumed in large quantities due to the presence of solanine. Over 90% of eggplant is concentrated in seven countries including China, India, Egypt, Turkey, and Japan (Lucier and Jernado, 2006). In Nigeria, eggplant is grown more in the South-Eastern parts of the country, especially in Abia State. It served alongside kola nuts in ceremonies (Okafor, 1993).

1.3.4 MEDICINAL IMPORTANCE OF EGGPLANT

The fruits of eggplant are processed and used in the preparation of condiments and products used in treating different diseases and health problems (Burkilll, 1985). A meal of garden egg is proven to be of benefits to patients suffering from raised intraocular pressure (glaucoma) and convergence insufficiency, as well as in heart diseases and Arteriosclerosis (Harish et al., 2008). The plant can be regarded as a portion of brain food because it houses the anthocyanin phytonutrient found in its skin. Nasunin, a potent antioxidant and free radical scavenger that has been shown to protect cell membranes from damage and fighting cancer and aging (Sabo and Dia, 2009). Studies have shown that nasunin protects fats in brain cell membranes. Iron is necessary for oxygen transport, normal immune function, and collagen synthesis but when it becomes too much in the bloodstream it becomes a major concern. Excess iron increases free radical production and is associated with an increased risk of heart disease and cancer. The chlorogenic acid performs antimutagenic (anti-cancer) activities in the body. It also performs anti- LDL (bad cholesterol) activities by increasing the levels of HDL (good cholesterol) in the body and at the same time has antiviral and antimicrobial properties.

1.3.5 ECONOMIC IMPORTANCE

The importance of the garden egg cannot be overemphasized because it is of global importance and consumed on daily basis by urban families and also represents the main source of income for producing households in West Africa (Danquah-Jones, 2000). At present eggplant is ranked as the third most important crop from the Solanaceae family after potato and tomato with a world production of 32 million tonnes. The greatest producers are China (18 million tons), India (8.4 million tons) followed by Egypt (1 million tons), and Turkey (0.8 million tons). Italy (0.3 million tons) and Spain (0.2 million tons) are important eggplant producers in Europe (FAOSTAT, 2007).

1.6 PRODUCTION OF EGGPLANT

Although commonly sold in the American, European, and Australian markets, over 90 percent of eggplant production is concentrated in seven countries which include China, India Egypt, Turkey, and Japan (Lucier and Jernado, 2006). In 2007, the world production of eggplant was estimated to be 32 million tons, with China and India as leading producers of 18 million tons and 8.4 million tons respectively, followed by Egypt (1million tons) and Turkey (0.8 million tons). Also, Italy and Spain, as the leading eggplant producers in Europe, produced 0.3 million tons and 0.2 million tons respectively (FAOSTAT, 2007).

In 2016, the global production of eggplants was 51.3million tonnes. That year, almost 1.8million hectares (4.4million acres) were devoted to the cultivation of eggplants in the world. Over 62% of that output came from China alone, India (24.5% of world total), Egypt, Turkey, and Iran were also major producers.

TABLE 1.2: Top Countries in Eggplant Production 2016

1.3.7 PRODUCTION OF THE CONSTRAINTS OF GARDEN EGG

The production of eggplant is affected by several factors ranging from diseases and pests, nutrient deficiency, availability of inputs, and seed availability. However, the main production constraints are as follows:

DISEASE AND PEST

According to Onuoha (2005) and Maraizu (2007), eggplant is faced with a lot of challenges which include pests and diseases during production, distribution of the product to areas of needs, and storage. Pests and diseases affect the crop by reducing total production as well as product quality. The shoot and fruit borer (SFB), Leucinodes orbonalis), is one of the main pest problems. SFB is widely distributed across all areas in Nigeria that produce eggplant. The damage caused by the pest affects the price significantly in local markets. Ramatu et al., (2007) reported that the yield losses from SFB could be as high as 21%, still, the damaged fruits are sold at discounted prices. This pest reduces productivity and chances of having net returns below Zero. Eggplant production is profitable under the use of pesticides as it increases the yield, net return, and revenue of the farmers in the study area.

SEED AVAILABILITY

This is a major constraint in production because farmers find it difficult to get good and quality seeds which have reduced productivity. But with institutions like the International Institute of Tropical Agriculture (IITA) and centers like the National Center for Genetic Resources and Biotechnology (NACGRAB) which have improved on conservation of seeds, farmers can now easily get good and quality seeds for production.

1.4 PROBLEM STATEMENT

In Nigeria, eggplant farm yields and productivity remain a challenge for millions of smallholder farmers, and an important component of this is seeds. The high demand for seeds has affected the quality of seeds sold in the market. Eggplant productivity is greatly impacted by the sale and use of fake or adulterated seeds.  Various seed dealers involved in the sale of adulterated seeds to unsuspecting farmers are a big challenge to the seed industry. Their activities are a disservice to the nation, as they rob unsuspecting farmers of their increase in productivity and further make farmers have a wrong impression of improved and good quality seeds. This is further complicated by the fact that there is little or non-existent eggplant germplasm to conserve the genetic purity of eggplant varieties currently available in the country.

Another important problem faced by eggplant breeding programs, as well as in other species, is a narrowing of the genetic base. To create variations, time-consuming and expensive methods are needed, including mutation breeding, interspecific hybridization, and biotechnological approaches (Boyaci et al., 2015).

1.5 JUSTIFICATION.

Genetic characterization of the DNA of plants is one of the best ways of conserving the genetic purity of the plants’ germplasm (Papa and Gepts, 2003). Generating genetic information of eggplant in Nigeria will not only be useful in conservation but will be useful in the eggplant improvement programs of mandate research institutions such as the National Horticultural Research Institute (NIHORT) and National Center for Genetic Resource and Biotechnology (NACGRAB). Therefore, the collection and characterization of genetic resources is required for the improvement of new varieties (Boyaci et al., 2015).

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